Three tRNAs were labeled with 75Se in cultures of Clostridium sticklandii incubated in the presence of 75SeO3 and antibiotics that block either protein synthesis or DNA-dependent RNA synthesis. These tRNAs, tentatively called seleno-tRNAs I, II, and III according to their elution sequence from benzoylated DEAE-cellulose column, were partially purified by chromatography on benzoylated DEAE-cellulose and DEAE-Sephadex A-50 columns. The 75Se in these tRNAs was in the polynucleotide portion of the molecule and not in the form of esterified (alkali-labile) selenocysteine. The purified seleno-tRNA II could be aminoacylated with L-proline. The L-proline acceptor activity was cochromatographed with the seleno-tRNA II on the DEAE-Sephadex A-50 column. These results suggest that seleno-tRNA II is a selenium-containing L-prolyl-tRNA.